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Abstract

The
year 2017 marks the twentieth anniversary of terpenoid cyclase
structural biology: a trio of terpenoid cyclase structures reported
together in 1997 were the first to set the foundation for understanding
the enzymes largely responsible for the exquisite chemodiversity of
more than 80000 terpenoid natural products. Terpenoid cyclases catalyze
the most complex chemical reactions in biology, in that more than
half of the substrate carbon atoms undergo changes in bonding and
hybridization during a single enzyme-catalyzed cyclization reaction.
The past two decades have witnessed structural, functional, and computational
studies illuminating the modes of substrate activation that initiate
the cyclization cascade, the management and manipulation of high-energy
carbocation intermediates that propagate the cyclization cascade,
and the chemical strategies that terminate the cyclization cascade.
The role of the terpenoid cyclase as a template for catalysis is paramount
to its function, and protein engineering can be used to reprogram
the cyclization cascade to generate alternative and commercially important
products. Here, I review key advances in terpenoid cyclase structural
and chemical biology, focusing mainly on terpenoid cyclases and related
prenyltransferases for which X-ray crystal structures have informed
and advanced our understanding of enzyme structure and function.

Taxol (paclitaxel) is a potent anticancer drug first isolated from the Taxus brevifolia Pacific yew tree. Currently, cost-efficient production of Taxol and its analogs remains limited. Here, we report a multivariate-modular approach to metabolic-pathway engineering that succeeded in increasing titers of taxadiene--the first committed Taxol intermediate--approximately 1 gram per liter (~15,000-fold) in an engineered Escherichia coli strain. Our approach partitioned the taxadiene metabolic pathway into two modules: a native upstream methylerythritol-phosphate (MEP) pathway forming isopentenyl pyrophosphate and a heterologous downstream terpenoid-forming pathway. Systematic multivariate search identified conditions that optimally balance the two pathway modules so as to maximize the taxadiene production with minimal accumulation of indole, which is an inhibitory compound found here. We also engineered the next step in Taxol biosynthesis, a P450-mediated 5α-oxidation of taxadiene to taxadien-5α-ol. More broadly, the modular pathway engineering approach helped to unlock the potential of the MEP pathway for the engineered production of terpenoid natural products.

Streptomycetes are high G+C Gram-positive, antibiotic-producing, mycelial soil bacteria. The 8.7-Mb Streptomyces coelicolor genome was previously sequenced by using an ordered library of Supercos-1 clones. Here, we describe an efficient procedure for creating precise gene replacements in the cosmid clones by using PCR targeting and lambda-Red-mediated recombination. The cloned Streptomyces genes are replaced with a cassette containing a selectable antibiotic resistance and oriT(RK2) for efficient transfer to Streptomyces by RP4-mediated intergeneric conjugation. Supercos-1 does not replicate in Streptomyces, but the clones readily undergo double-crossover recombination, thus creating gene replacements. The antibiotic resistance cassettes are flanked by yeast FLP recombinase target sequences for removal of the antibiotic resistance and oriT(RK2) to generate unmarked, nonpolar mutations. The technique has been used successfully by >20 researchers to mutate around 100 Streptomyces genes. As an example, we describe its application to the discovery of a gene involved in the production of geosmin, the ubiquitous odor of soil. The gene, Sco6073 (cyc2), codes for a protein with two sesquiterpene synthase domains, only one of which is required for geosmin biosynthesis, probably via a germacra-1 (10) E,5E-dien-11-ol intermediate generated by the sesquiterpene synthase from farnesyl pyrophosphate.